Approximately 170 million people worldwide are infected persistently with hepatitis C virus (HCV) and these individuals account for most cases of chronic liver disease (Wasley and Alter, 2000). The public health impact of HCV is compounded by the low response rate to interferon (IFN)-based therapies, underscoring the need for new therapeutic strategies and new drug targets (McHutchison and Patel, 2002).
HCV is a single-stranded positive-sense RNA virus of the Flaviviridae family (Reed and Rice, 1998). The 9.6-kilobase HCV genome encodes a single polyprotein that is post-translationally processed into at least 10 structural and nonstructural (NS) proteins. Among the nonstructural (NS) proteins, NS3, NS4A, NS4B, NS5A, and NS5B are sufficient to support replication of the HCV RNA (Lohmann et al., 1999). Current studies support a model in which HCV infection results in assembly of the viral RNA and NS proteins into a replication complex that associates with cellular membranes, most likely a modified endoplasmic reticulum (Egger et al., 2002; El-Hage and Luo, 2003). The cellular proteins required for assembly and maintenance of the HCV replication complex are not known.
Inasmuch as native HCV cannot be efficiently propagated in cultured cells (Reed and Rice, 1998), genome-length and subgenomic HCV RNA replicons have been developed to facilitate the study of viral RNA replication. These HCV RNA replicon systems encompass either the entire HCV genome or only the NS3-5B protein coding region within a neomycin-selectable, bicistronic RNA. When introduced into human hepatoma (Huh7) cells, the HCV replicon RNA replicates autonomously on intracellular membranes (Lohmann et al., 1999; Egger et al., 2002).
We recently reported that HCV RNA replication in Huh7 cells can be disrupted by treatment with lovastatin, a drug that decreases the production of mevalonate by inhibiting 3-hydroxy-3-methylglutaryl CoA reductase (Ye et al., 2003). Mevalonate is a precursor of two hydrophobic prenyl groups, farnesyl (15 carbons) and geranylgeranyl (20 carbons), which are attached to various cellular proteins, anchoring them to membranes (Goldstein and Brown, 1990). Inhibition of HCV RNA replication by lovastatin was overcome by the addition of geranylgeraniol, but not farnesol, suggesting that HCV RNA replication requires one or more geranylgeranylated proteins (Ye et al., 2003). Kapadia and Chisari (2005) have subsequently reported similar results.
The role for a geranylgeranylated protein in HCV RNA replication is further supported by our additional finding that the replication could be blocked by an inhibitor of geranylgeranyl transferase I (GGTase-I) (Ye et al., 2003), an enzyme that transfers geranylgeranyl groups to many cellular proteins (Seabra et al., 1991; Zhang and Casey, 1996). GGTase-I attaches geranylgeranyl in thioether linkage to cysteine residues in proteins that contain a COOH-terminal Cys-A-A-X sequence (CAAX box), where C is cysteine, A is an aliphatic amino acid, and X is typically leucine (or rarely isoleucine, valine, or phenylalanine) (Reid et al., 2004).
Prenylated proteins can be labeled by incubating cultured cells with [3H]mevalonate, which is enzymatically converted to [3H]farnesyl pyrophosphate and [3H]geranylgeranyl pyrophosphate, the donors in the protein prenylation reactions. Prenylated proteins within the 3H-labeled cell extracts can then be analyzed by SDS-PAGE and [3H]autoradiography. Labeling with [3H]mevalonate can be dramatically improved through the use of Met-18b-2 cells, a line of mutant Chinese hamster ovary (CHO) cells that takes up mevalonate at a very high rate (Faust and Krieger, 1987), owing to a gain-of-function point mutation in a monocarboxylate transporter (Garcia et al., 1994).
We used a combination of [3H]mevalonate labeling, co-immunoprecipitation, and bioinformatic search to identify a geranylgeranylated host protein required for HCV RNA replication. We show that this protein, called FBL2, forms a specific complex with the HCV NS5A protein and that the FBL2-NS5A complex is crucial for HCV RNA replication. FBL2 is a previously identified protein that belongs to the family of F-box proteins and was originally cloned based on its homology to Skp2 (S-phase-kinase-associated protein 2), one of the best characterized proteins in this family (Ilyin et al., 1999). Like other F Box-containing proteins, FBL2 contains two distinct motifs: 1) an NH2-terminal F-box that mediates the interaction between F-box proteins and the SCF E3 ubiquitin ligase complex (Cardozo and Pagano, 2004); and 2) 11 leucine-rich repeats involved in protein-protein interaction (Kobe and Kajava, 2001). Unlike other F-box proteins, FBL2 contains a potential geranylgeranylation sequence, CVIL, at the COOH-terminus. FBL2 is widely expressed in multiple tissues, including the liver (Ilyin et al., 1999).